Anti-microbial agents

ABSTRACT

Antimicrobial agents that can serve as replacements to conventional pharmaceutical antibiotics are disclosed. The antimicrobial agents comprise conjugatively transmissible plasmids that kill targeted pathogenic bacteria, but are not harmful to donor bacteria. Two types of lethal transmissible plasmids are disclosed. One type kills recipient bacteria by unchecked (“runaway”) replication in the recipient cells and is prevented from occuring in donor cells. Another type kills recipient bacteria by expressing a gene that produces a product detrimental or lethal to recipient bacterial cells, that gene being prevented from expression in donor cells.

[0001] This application claims priority to U.S. application Ser. No.09/651,290, filed Aug. 30, 2000, the entirety of which is incorporatedby reference herein.

[0002] Pursuant to 35 U.S.C. §202(c), it is acknowledged that the UnitedStates government has certain rights in the invention described herein,which was made in part with finds from the National Institutes ofHealth, Grant No. GM40314.

FIELD OF THE INVENTION

[0003] The present invention relates to the field of bacteriology. Inparticular, the invention relates to novel antimicrobial agentscomprising transmissible plasmids that kill targeted recipient bacteria,but are not harmful to donor bacteria.

BACKGROUND OF THE INVENTION

[0004] Various patents, patent publications and scientific articles arereferenced in parentheses throughout the specification. Thesepublications are incorporated by reference herein in their entireties.

[0005] As the use of conventional pharmaceutical antibiotics (hereinreferred to as antibiotics) increases for medical, veterinary andagricultural purposes, the increasing emergence of antibiotic-resistantstrains of pathogenic bacteria is an unwelcome consequence. This hasbecome of major concern inasmuch as drug resistance of bacterialpathogens is presently the major cause of failure in the treatment ofinfectious diseases. Indeed, people now die of certain bacterialinfections that previously could have been easily treated with existingantibiotics. Such infections include, for instance, Staphylococcuspneumoniae, causing meningitis; Enterobacter sp., causing pneumonia;Enterococcus sp., cawing endocarditis, and Mycobacterium tuberculosis,causing tuberculosis.

[0006] The emergence of single- or multi-drug resistant bacteria resultsfrom a gene mobilization that responds quickly to the strong selectivepressure that is a consequence of antibiotic uses. Over the last severaldecades, the increasingly frequent usage of antibiotics has acted inconcert with spontaneous mutations arising in the bacterial gene pool toproduce antibiotic resistance in certain strains. This gene pool iscontinually utilized by previously sensitive strains capable ofaccessing it by various means including the transfer of extrachromosomalelements (plasmids) by conjugation. As a result, single- and multi-drugresistance genes are commonly found in a large variety of bacterialplasmids.

[0007] Presently there is no known method by which to avoid theselection of antibiotic resistant bacterial mutants that arise as aresult of the many standard applications of antibiotics in the modernworld. Accordingly, a need exists to develop alternative strategies ofantibacterial treatment.

[0008] Interest in the use of bacteriophages to treat infectiousbacterial diseases developed early in the twentieth century, and hasundergone a resurgence in recent years. For instance, bacteriophageshave been shown effective in the treatment of certain pathogenic E. colispecies in laboratory and farm animals, and have been proposed as aviable alternative to the use of antibiotics (Smith & Huggins, J. Gen.Microbiol. 128: 307-318, 1981; Smith & Huggins, J. Gen. Microbiol. 129:2659-2675, 1983; Smith et al., J. Gem Microbiol. 133: 1111-1126, 1986;Kuvda et al., Appl. Env. Microbiol. 65: 3767-3773, 1999). However, theuse of bacteriophages as antimicrobial agents has certain limitations.First, the relationship between a phage and its host bacterial cell istypically very specific, such that a broad host-range phage agentgenerally is unavailable. Second, the specificity of interaction usuallyarises at the point of the recognition and binding of phage to the hostcell. This often occurs through the expression of surface receptors onthe host cell to which a phage specifically binds. Inasmuch as suchreceptors are usually encoded by a single gene, mutations in the hostbacterial cell to alter the surface receptor, thereby escaping detectionby the phage, can occur with a frequency equivalent to or higher than,the mutation rate to acquire antibiotic resistance. As a result, ifphage were utilized as commonly as antibiotics, resistance of pathogenicbacteria to phages could become as common a problem as antibioticresistance.

[0009] Another approach to controlling pathogenic bacteria has beenproposed, which relies on using molecular biological techniques toprevent the expression of antibiotic resistance genes in pathogenicbacteria (U.S. Pat. No. 5,976,864 to Altman et al.). In this method, anucleic acid construct encoding an “external guide sequence” specificfor the targeted antibiotic resistance gene is introduced into thepathogenic bacterial cells. The sequence is expressed, hybridizes withmessenger RNA (mRNA) encoding the antibiotic resistance gene product,and renders such mRNA sensitive to cleavage by the enzyme RNAse P. Sucha system also has limited utility, since it is targeted to specificantibiotic resistance genes. While the system may be effective inovercoming resistance based on expression of those specific genes,continued use of the antibiotics places selective pressure on thebacteria to mutate other genes and develop resistance to the antibioticby another mechanism.

[0010] It is clear from the foregoing discussion that currentalternatives to antibiotic use are limited and suffer many of the samedrawbacks as antibiotic use itself. Thus, a need exists for a method ofcontrolling unwanted bacteria that is flexible in range and that cannotbe overcome by the bacteria by a single or small number of mutations.

SUMMARY OF THE INVENTION

[0011] The present invention provides novel antibacterial agents thatare efficiently transferred to bacteria, e.g., pathogenic bacteria, thathave a flexible range, and to which the target bacteria have difficultydeveloping resistance. These antibacterial agents offer an effectivealternative to the use of conventional antibiotics. This inventionrelies on a universal property of conjugative systems wherebyplasmid-encoded information, even that encoding self-destruction, willbe expressed upon transfer to a recipient cell. That property was usedto engineer a broad-host lethal system to limit the lateral spread ofcloned genes (Diaz et al., 1994, Mol. Microbiol. 13, 855-861).

[0012] According to one aspect of the invention, an antibacterial agentis provided that comprises a donor cell, e.g., a non-pathogenicbacterial cell, harboring at least one transmissible plasmid having thefollowing features: (a) an origin of replication for synthesizing theplasmid DNA in the donor cell, wherein initiation of replication at theorigin of replication is negatively controlled by a plasmid replicationrepressor; (b) an origin of transfer to provide the initiation site forconjugative transfer of the transmissible plasmid from the donor cell toat least one recipient cell; and (c) at least one selectable markergene. The donor cell further comprises one or more conjugative transfergenes conferring upon the donor cell the ability to conjugativelytransfer the transmissible plasmid to the recipient cell. The donor cellalso produces the plasmid replication repressor. In some embodiments,the recipient cell is a bacterium that does not produce the plasmidreplication repressor. In preferred embodiments, the recipient cell ispathogenic.

[0013] According to another aspect of the invention, an antibacterialagent is provided which comprises a donor cell, e.g., a non-pathogenicbacterial cell, harboring at least one transmissible plasmid comprisingthe following features: (a) an origin of replication for synthesizingthe plasmid DNA in the donor cell; (b) an origin of transfer to providethe start site for conjugative transfer of the transmissible plasmidfrom the donor cell to at least one recipient cell; and (c) at least onekiller gene that, upon expression in a recipient cell, produces aproduct that kills the cell. The donor cell again comprises one or moretransfer genes conferring upon the donor cell the ability toconjugatively transfer the transmissible plasmid to the recipient cell,and is modified so as to be unaffected by the product of the killergene. In some embodiments,the recipient cell is a bacterium thatisaffected by the product of the killer gene. In preferred embodiments,the recipient cell is pathogenic.

[0014] The present invention also provides methods of treating a subjectfor a bacterial infection, which comprises administering to the subjectone of the aforementioned antibacterial agents. A mode of administrationis selectedsuch that the donor cells of the antibacterial agent comeinto conjugative proximity to the unwanted recipientcells, such that thetransmissible plasmids of the donor cells are conjugatively. transferredfrom the donors to the unwanted recipient cells. In some embodiments,the transferred plasmidundergoes unchecked replication. In otherembodiments, at least one killer gene is expressed to produce a geneproduct that is detrimental or lethal to the unwanted recipientcells.

[0015] The present invention also provides pharmaceutical preparationsfor treating a patient for a bacterial infection. These preparationscomprise one of the aforementioned antibacterial agents, formulated fora pre-determined route of administration to the patient.

[0016] The present invention further provides methods of using theantibacterial agents of the invention in agricultural, veterinary,environmental and food maintenance applications. In these methods, theantibacterial agents of the invention are applied to (1) plant surfacesto reduce or prevent bacterial plant disease or spoilage, (2) foodsurfaces to reduce or prevent post harvest spoilage of vegetables, meator fish, (3) animal feed to reduce the bio-burden. Other similarapplications are also provided.

[0017] Other features and advantages of the present invention will beunderstood by reference to the drawings, detailed description andexamples that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1. Schematic diagram showing a process of killing bacteria byconjugative transfer of plasmids that engage in runaway replication inthe recipient cells.

[0019]FIG. 2A. Schematic diagram of a non-self-transmissible, runawayreplication plasmid system using a helper plasmid and a transmissiblerunaway replication plasmid.

[0020]FIG. 2B. Schematic diagram of a self-transmissible, runawayreplication plasmid system.

[0021]FIG. 3. Schematic diagrams showing a “Trojan-Horse”-like processof killing bacteria by conjugative transfer of plasmids that encode akill product that is neutralized by an anti-kill product in the donorbut is not neutralized in the recipient that lacks anti-kill gene aspart of its chromosome (top). Bottom diagram represents a generalscenario of process of killing bacteria by conjugatively transferredplasmid that contains a synthetically assembled operon that encodes oneor more kill products. Expression of the operon is repressed in thedonor but not in the recipient. The kill gene products can be eithernatural or man-made peptides or RNA.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention provides novel antibacterial strategiesthat utilize the highly efficient bacterial conjugation system totransfer a “killer” plasmid from a donor cell that is engineered to beresistant to the killer plasmid, to a target bacterial cell that is not.

[0023] In one aspect of the invention, the “killer plasmid” is one thatundergoes runaway replication in the recipient cells, ultimately killingthe cells. The basic principles underlying the mechanism by whichrunaway plasmid replication kills cells are outlined below.

[0024] Plasmids are generally dispensable DNA molecules that are stablymaintained in bacterial populations. Plasmids replicateextra-chromosomally inside the bacterium and can transfer their DNA fromone cell to another by a variety of mechanisms. DNA sequencescontrolling extra chromosomal replication (ori) and transfer (tra) aredistinct from one another; i.e., a replication sequence generally doesnot control plasmid transfer, or vice-versa. Replication and transferare both complex molecular processes that make use of both plasmid- andhost-encoded functions.

[0025] Bacterial conjugation is the unidirectional and horizontaltransmission of genetic information from one bacterium to another. Thegenetic material transferred may be a plasmid or it may be part of achromosome. Bacterial cells possessing a conjugative plasmid contain asurface structure (the sex pilus) that is involved in the coupling ofdonor and recipient cells, and the transfer of the genetic information.Conjugation involves contact between cells, and the transfer of genetictraits can be mediated by many plasmids.

[0026] Among all natural transfer mechanisms, conjugation is the mostefficient. For example, F plasmid of E. coli, pCF10 plasmid ofEnterococcus faecalis and pXO16 plasmid of Bacillus thuringiensis employdifferent mechanisms for the establishment of mating pairs, the sizes ofmating aggregates are different, and they have different host rangeswithin gram-negative (F) as well as gram-positive (pCF10 and pXO16)bacteria. Their plasmid sizes are also different; 54, 100 and 200 kb,respectively. Remarkably, however, those conjugation systems have veryimportant characteristics in common: they are able to sustainconjugative transfer in liquid medium and transfer efficiencies close to100% are often reached in a very short time (Dunny et al., 1982, J.Bacteriol. 151, 855-859; Andrup, et al., 1998, Plasmid 40, 30-43; AndrupL, and Andersen, K., 1999), Microbiology 145, 2001-2009; and Jansen etal., 1996, Curr. Microbiol. 33, 228-236. Thus, the conjugative processpermits the protection of plasmid DNA against environmental nucleases,and the very efficient delivery of plasmid DNA into a recipient cell.

[0027] Conjugation functions are plasmid encoded. Numerous conjugativeplasmids (and transposons) are known, which can transfer associatedgenes within one species (narrow host range) or between many species(broad host range). Transmissible plasmids have been reported innumerous Gram-positive genera, including but not limited to pathogenicstrains of Streptococcus, Staphylococcus, Bacillus, Clostridium andNocardia. The early stages of conjugation generally differ inGram-negative and Gram-positive bacteria. The role of some of thetransfer genes in conjugative plasmids from Gram-negative bacteria is toprovide pilus-mediated cell-to-cell contact, formation of a conjugationpore and related morphological functions. The pili do not appear to beinvolved in initiating conjugation in Gram-positive bacteria The featurebest understood in the Enterococci is the involvement of pheromones.Pheromones are hydrophobic polypeptides of 7-8 amino acids produced bypotential recipient cells. Pheromones invite attention of potentialdonor cells containing conjugative plasmids. PAD1 is one of the beststudied pheromone-indueed plasmids which can replicate in 50 differentbacterial hosts in addition to Enterococcus faecelis strains from whichit was initially isolated (Clewell D. B. 1999. Sex pheromone systems inEnterococci, In: Cell-Cell Signaling in Bacteria, Ed. G. M. Dunny, S. C.Winans; ASM, Washington D.C. pp 47-65). Moreover, conjugation can occurbetween genera as widely diverse as anaerobes and aerobes.

[0028] Naturally occurring plasmids are present within host cells at acharacteristic concentration (referred to herein as a particular plasmid“copy number”). Of great significance to the present invention is thefact that plasmid copy number is negatively controlled Helinski et al.,1996 (In Escherichia coli and Salmonella Cellular and Molecular Biology,Vol. 2 (ed F. Neidhardt, et al., 2295-2324, ASM Press, Washington D.C.).Thus, mutations that destroy the elements of the negative control causean over-replication phenotype that manifests itself by an increase inthe plasmid copy number (“copy-up” phenotype). In extreme cases ofcopy-up mutations, plasmid replication is completely unchecked due tothe loss of copy control mechanisms. This is referred to as “runawayplasmid replication” or simply “runaway replication.”

[0029] Runaway plasmid replication is lethal for the host cell. This isbecause, although the plasmid encodes the replication (Rep) protein thatcontrols its copy number, all other replication proteins are encoded bychromosomal genes. These chromosomally encoded proteins assemble into acomplex called a replisome. A typical bacterial cell possesses a small,fixed number of replisomes. Because both the chromosome and the plasmidsrequire the same replisomes for DNA synthesis, an excess of plasmidsacts like a trap to occupy all of the replisomes within the cell. Thisresults in the inability of the chromosome to replicate, ultimatelyleading to the death of the cell.

[0030] The use of runaway replication plasmids as a means to killrecipient cells has a number of advantages over conventional antibioticmethodologies. One significant advantage is that, since the entire hostreplication machinery is targeted, multiple mutations would be requiredto avoid death by elevating the expression or activity of the replisomesub-assemblies. For instance, mutations in ten genes would be requiredjust to increase the amount or activity of DNA polymerase III holoenzyme(composed of ten different subunits), and this polymerase is just one ofthe replisome sub-assemblies. Thus, there is little or no chance of abacterium acquiring resistance to being killed by over-replicatingplasmids. In contrast, conventional antibiotics typically inhibit only asingle enzymatic activity that is essential for the survival of a cell.A single-target strategy and the relatively high spontaneous mutationfrequency for one gene (10⁻⁶ to 10⁻⁸) unavoidably leads to the quickacquisition of resistance to such drugs.

[0031] Because runaway replication mutations are lethal to the hostcell, the donor cells that maintain such plasmids are generallyengineered so that replication of the plasmid is controlled e.g., byproviding a wild-type Rep protein to the host cell. In some embodiments,this is accomplished by providing a Rep gene on another plasmid. Inother embodiments, a rep gene is providedby integration into thebacterial chromosome of a donor cell using standard homologousrecombination techniques.

[0032] In some embodiments, the antimicrobial strategy of the presentinvention comprises:

[0033] (1) a plasmid that, alone or with the assistance of a helperplasmid, comprises the genes necessary to effect conjugative transfer ofthe plasmid from a donor cell to a recipient cell, wherein replicationof the plasmid is controlled, e.g., repressed, by a reversiblemechanism, such as control by a product of a gene that can bede-activated (e.g., via mutation) so as to release the control onplasmid replication (referred to herein as a “runaway replicationplasmid”);

[0034] (2) a source of conjugative transfer genes (e.g. on the runawayreplication plasmid, or on a separate “helper” plasmid); and

[0035] (3) a donor cell for maintaining the runaway replication plasmidin a replication-suppressed state, so as not to be killed by theplasmid.

[0036] A number of plasmids have been well characterized, and can serveas subjects for mutagenesis to create runaway mutants, which may be usedin embodiments of the present invention Such mutant plasmids contain, orcan be easily modified to contain all components needed for conjugativetransfer from donor to recipient cells but are defective in theirreplicative repressor (Rep) function. Examples of such mutants, bothbroad-range and narrow-range, are known in the art (Haugan et al.,Plasmid 33: 27-39, 1995; Molin et al., J. Bacteriol. 143: 1046-1048,1980; Toukdarian & Helinski, Gene 223: 205-211, 1998). A particularlypreferred plasmid of this type is a mutant of plasmid R6K, as describedin detail in Examples 1 and 2. Other examples include, but are notlimited to, RK2, pCU1, p15A, pIP501, pAMβ1 and pCRG1600.

[0037] As an alternative to the use of mutants, it may sometimes bepreferable to use various components of conjugative plasmids whosefeatures are well understood, to create plasmids having all necessaryfeatures. Such runaway replication plasmids or helper plasmids mayinclude (1) an origin of replication (e.g., oriV as decribed herein), asequence from which replication of the plasmid originates and thesequence that may be negatively regulated by a Rep protein; (2) anorigin of transfer (e.g., oriT as described herein), a sequence fromwhich a conjugal plasmid transfer originates; (3) transfer (tra) genesto effect conjugation; and (4) a screenable/selectable marker gene. Thedonor cell containing the runaway replication plasmid is engineered tocontain a functional repressor (Rep) of replication at oriV, therebycontrolling replication of the runaway replication plasmid while it isstill in the donor.

[0038] Non-self-transmissible plasmid systems and self-transmissibleplasmid systems are contemplated. Examples of these are shownschematically in FIGS. 2A and 2B. The systems diagrammed here anddescribed below are provided as examples of the systems of the presentinvention and are not be construed as limiting the components or sourcesof components assembled to effect the methods and compositions of theinvention. For example, where particular genes or genetic elementsproviding particular functions are named (e.g., the oriV origin ofreplication, the oriT origin of transfer), it is contemplated that othergenes or genetic elements providing equivalent functions or functionalcombinations may be used.

[0039] In some embodiments of non-self-transmissible systems (e.g., asshown in FIG. 2A), the runaway replication plasmid contains an oriT, anoriV and a selectable marker gene. In some embodiments, a helper plasmidcontains the additional tra genes, along with its own origin ofreplication and a selective marker. Thus, the helper plasmid enablesconjugative transfer of the runaway replication plasmid, but is itselfconfined to the donor cell due to its lack of an oriT. In otherembodiments, the tra genes are integrated into the chromosome of thedonor cell. Since the runaway replication plasmid lacks the necessarytra genes to convert the recipient cell into a donor cell, the cycle ofconjugation ends with the original recipient cell. It cannot transferits runaway replication plasmid to a second recipient before it dies.

[0040] In some embodiments of self-transmissible systems (e.g., as shownin FIG. 2B), the runaway replication plasmid contains an oriT, an oriVand a selectable marker gene. It also contains the additional tra genesneeded for conjugative transfer. Thus, unlike the non-self-transmissibleplasmid described above, once this plasmid has been transmitted from theoriginal donor to a first recipient, it is capable of transmittingitself again to subsequent recipients before the first recipient cell iskilled by runaway replication. A plasmid of this type has the capabilityof much faster dissemination among recipient cells than thenon-self-transmissible type, resulting in faster and more widespreadkilling of those cells.

[0041] In either the self-transmissible or the self-non-transmissiblesystem, the donor cells generally comprise a means of controllingplasmid replication. In some embodiments, the control comprises a geneencoding a repressor of plasmid replication. For example, the Repprotein represses plasmid replication initiated at oriV. In someembodiments, a Rep-encoding gene is provided on a helper plasmid. Inother embodiments, a Rep-encoding gene is integrated into the donor cellchromosomal DNA. Plasmid DNA comprising the Rep-encoding gene may beintroduced into bacterial cells by any commonly known technique (e.g.,transformation). The Rep-encoding gene can be integrated into the hostgenome by a site-specific recombination, according to standard methods(Li-Ch Huang, E. Wood and M. Cox; J. Bacteriol. 179: 6076-6083, 1997).

[0042] A number of bacterial oriV's and the Rep proteins that negativelycontrol them have been characterized. Each of these is contemplated foruse in the present invention. Examples of suitable oriV/Rep systems foruse in the invention include, but are not limited to: RK2, R6K, rts 1,p15A, RSF1010, F and P1. A wide variety of replication systems may beused in the present invention (see, e.g., U.S. Pat. No. 5,851,808). Thepresent invention is not limited to those systems described above.

[0043] The selection of oriV will confer on the system its range ofpotential recipients for runaway replicating plasmids. In most instancesit may be preferable to target a specific recipient of the runawayreplication plasmid. Such instances include, but are not limited tousing the conjugative runaway plasmids for combating Enterobacteria,Enterococci, Staphylococci and non-sporulating Gram-positive pathogenssuch as Nocardia and Mycobacterium sp. Examples of selective host rangeplasmids from which such oriV's may be obtained include, but are notlimited to, P1 and F.

[0044] In instances where it is desirable to affect a wide variety ofrecipients, a broad range oriV is employed. Examples of broad range(“promiscuous”) plasmids from which oriVs may be obtained include, butare not limited to: R6K, RK2, p15A and RSF1010.

[0045] As used herein, the term “range” (or “host range”) refersgenerally to parameters of both the number and diversity of differentbacterial species in which a particular plasmid (natural or recombinant)can replicate. Of these two parameters, one skilled in the art wouldconsider diversity of organisms as generally more defining of hostrange. For instance, if a plasmid replicates in many species of onegroup, e.g., Enterobacteriaceae, it may be considered to be of narrowhost range. By comparison, if a plasmid is reported to replicate in onlya few species, but those species are from phylogenetically diversegroups, that plasmid may be considered of broad host range. As discussedabove, both types of plasmids (or components thereof) will find utilityin the present invention.

[0046] Conjugative transfer (tra) genes also have been characterized inmany conjugative bacterial plasmids. The interchangeability between thegene modules conferring the ranges of hosts susceptible for conjugaltransfer and vegetative replication include Gram-positive andGram-negative species. Examples of characterized tra genes that aresuitable for use in the present invention are the tra genes from: (1) F(Firth, N., Ippen-Ihler, K. and Skurray, R. A. 1996, Structure andfunction of F factor and mechanism of conjugation. In: Escherichia coliand Salmonella, Neidhard et al., eds., ASM Press, Washington D.C.); (2)R6K (Nunez et al., Mol. Microbiol. 24: 1157-1168, 1997); and (3) Ti(Ferrand et al., J. Bacteriol. 178: 4233-4247, 1996). Additional tragenes that find use with the present invention include, but are notlimited to, those described in U.S. Pat. Nos. 6,180,406 and 6,251,674.

[0047] According to another aspect of the invention, the bacterialconjugation system is again utilized, this time to efficiently deliver avariety of “killer genes” to target bacterial cells. The delivery ofvarious killer genes to bacterial cells occurs in nature, upon infectionof bacteria with bacteriophages. Bacteriophages utilize a number ofdifferent mechanisms to maintain their own replication cycles, generallyresulting in lysis of the host bacterial cells. Indeed, bacteriophageshave been proposed and used as alternatives to antibiotics, as discussedabove in the Background of the Invention. One serious drawback to theuse of bacteriophages for this purpose is that they are often extremelyhost-specific, binding only to cell surfaces possessing specificreceptors. As a result, bacteria quickly develop resistance mutations inthe receptor, thereby escaping recognition by the phage. The presentinvention circumvents that drawback by placing one or more killer genes(e.g., from a phage or other source) on a conjugative plasmid. Theconjugative plasmid containing the killer gene(s), like the conjugativerunaway replication plasmids described above, is thereafter efficientlydistributed to recipient cells, killing them shortly thereafter.Additional killing systems include, but are not limited to, thosedescribed in U.S. Pat. No. 6,277,608.

[0048] Bacteriophage kill host cells by a variety of mechanisms, many ofwhich are encoded by a discrete set of genes in the phage genome. Forinstance, bacteriophage MS2 contains a gene encoding a bacterial lysisprotein (Coleman et al., Bacteriol. 153: 1098-1100, 1983). Phage T4Dcontains genes encoding proteins that degrade cytosine-containing DNA inbacterial host cells (Kutter and Wilberg, J. Mol. Biol. 38: 395-411,1968). Other T4 phage encode gene products that interfere withtranscription of cytosine-containing DNA (Drivdahl and Kutter, J.Bacteriol. 172: 2716-2727, 1990). Yet other T4 gene products areresponsible for the disruption of the bacterial nucleoid (Bouet et al.,Gene 141: 9-16, 1994). Over 5000 characterized bacteriophages provideenormous reservoir of killer genes (Ackermann 2001. Arch. Vir., 146,843-857). Such killer genes can be inserted into a conjugative plasmidsuch as those described above, for efficient distribution to targetrecipient cells.

[0049] In addition, other types of killer genes may be utilizedsimilarly. These include naturally-occurring or synthetic genes. Anonlimiting example of a naturally-occurring gene that is suitable foruse in the invention is the hok gene product described by Gerdes et al.(EMBO J. 5: 2023-2029, 1986). Examples of man-made nucleic acidmolecules that may be used in this aspect of the invention include: (1)sequences encoding peptides with bactericidal activity and endotoxinneutralizing activity for Gram-negative bacteria as described in U.S.Pat. No. 5,830,860; (2) sequences encoding RNA molecules with bindingaffinity to critical bacterial cellular targets (e.g., Chen and Gold,Biochemistry 33: 8746-8756, 1994); and (3) oligonucleotides generated bythe SELEX method for the in vitro evolution of nucleic acid moleculeswith highly specific binding to target molecules as described in U.S.Pat. No. 5,475,096 and U.S. Pat No. 5,270,163.

[0050] In these systems, steps may be employed to prevent death of thedonor cell For example, the death of the donor cell can be prevented byemploying a synthetic promoter-operator system whose expression isprevented by the repressor made only in the donor cells (FIG. 3 bottom).In other embodiments, the toxin can be neutralized by an antitoxin madein donor but not in recipient bacteria (FIG. 3 top).

[0051] In preferred embodiments, the plasmid contains a screenable orselectable marker gene. In traditional molecular biologicalmanipulations of recombinant bacteria, the selectable marker gene is anantibiotic resistance gene. Since the present invention is designed toavoid further spread of antibiotic resistance, an alternative selectablemarker system is preferred for use in the present invention.Accordingly, though antibiotic resistance markers can be used inlaboratory tests, preferred selectable markers are nutritional markers,i.e., any auxotrophic strain (e.g., Trp⁻, Leu⁻, Pro⁻) containing aplasmid that carries a complementing gene (e.g., trp⁺, leu⁺, pro⁺).

[0052] The donor bacterial strain for any of the above-described killerplasmids can be any one of thousands of free-living bacteria, associatedwith the body of warm-blooded animals, including humansand plants.Preferably, non-pathogenic bacteria that colonize the non-sterile partsof the body (e.g., skin, digestive tract, urogenital region, mouth,nasal passages, throat and upper airway, ears and eyes) are utilized asdonor cells, and the methodology of the invention is used to combatbacterial infections of these parts of the body. In another embodiment,safe donors of these plasmids are developed for combating systemicinfection. Examples of particularly preferred donor bacterial speciesinclude, but are not limited to: (1) non-pathogenic strains ofEscherichia coli (E. coli F18 and E. coli strain Nissle 1917), (2)various species of Lactobacillus (such as L. casei, L. plantarum, L.paracasei, L. acidophilus, L. fernentum, L. zeae and L. gasseri), (3)other nonpathogenic or probiotic skin-or GI colonizing bacteria such asLactococcus, Bifidobacteria, Eubacteria, and (4) bacterial mini-cells,which are anucleoid cells destined to die but still capable oftransferring plasmids (see; e.g., Adler et al., Proc. Natl. Acad. Sci.USA 57; 321-326, 1970; Frazer and Curtiss III, Current Topics inMicrobiology and Immunology 69: 1-84, 1975; U.S. Pat. No. 4,968,619 toCurtiss III).

[0053] In some embodiments, the target recipient cells are pathogenicbacteria dispersed throughout the body, but particularly on the skin orin the digestive tract, urogenital region, mouth, nasal passages, throatand upper airways, eyes and ears. Of particular interest for targetingand eradication are pathogenic strains of Pseudomonas aeruginosa,Escherichia coli, Staphylococcus pneumoniae and other species,Enterobacter spp., Enterococcus spp. and Mycobacterium tuberculosis. Thepresent invention finds use with a wide array of target organisms (e.g.,pathogenic organisms), whether in therapeutic, agricultural, or othersettings, including, but not limited to, those described in U.S. Pat.Nos. 6,271,359, 6,261,842, 6,221,582, 6,153,381, 6,106,854, and5,627,275. Others are also discussed herein, and still others will bereadily apparent to those of skill in the art.

[0054] It is clear from the foregoing discussion that numerous types ofkiller plasmids (e.g., runaway replication plasmids, plasmids carryinglethal phage genes, etc.) are suitable for use in the present invention.In view of this, one of skill in the art will appreciate that a singledonor bacterial strain might harbor more than one type of killer plasmid(e.g., runaway or toxin-producing). In other embodiments, a donor cellmay harbor a killer plasmid expressing multiple kill functions, as shownin FIG. 3 (bottom) or may harbor multiple killer plasmids eachexpressing kill function(s) independently of the other plasmids. Thussuch multiple plasmid systems can contain a plurality of plasmid-encodedfunctions targeted to different recipient cells. Further, two or moredonor bacterial strains, each containing one or more killer plasmids,may be combined for a similar multi-target effect.

[0055] The systems of the present invention find utility for treatmentof humans and in a variety of veterinary, agronomic, horticultural andfood processing applications. For human and veterinary use, anddepending on the cell population or tissue targeted for protection, thefollowing modes of administration of the bacteria of the invention arecontemplated: topical, oral, nasal, pulmonary/bronchial (e.g., via aninhaler), ophthalmic, rectal, urogenital, subcutaneous, intraperitonealand intravenous. The bacteria preferably are supplied as apharmaceutical preparation, in a delivery vehicle suitable for the modeof administration selected for the patient being treated. The term“patient” or “subject” as used herein refers to humans or animals(animals being particularly useful as models for clinical efficacy of aparticular donor strain).

[0056] For instance, to deliver the bacteria to the gastrointestinaltract or to the nasal passages, the preferred mode of administration isby oral ingestion or nasal aerosol, or by feeding (alone or incorporatedinto the subject's feed or food). In this regard, it should be notedthat probiotic bacteria, such as Lactobacillus acidophilus, are sold asgel capsules containing a lyophilized mixture of bacterial cells and asolid support such as mannitol. When the gel capsule is ingested withliquid, the lyophilized cells are re-hydrated and become viable,colonogenic bacteria. Thus, in a similar fashion, donor bacterial cellsof the present invention can be supplied as a powdered, lyophilizedpreparation in a gel capsule, or in bulk for sprinkling into food orbeverages. The re-hydrated, viable bacterial cells will then populateand/or colonize sites throughout the upper and lower gastrointestinalsystem, and thereafter come into contact with the target pathogenicbacteria.

[0057] For topical applications, the bacteria may be formulated as anointment or cream to be spread on the affected skin surface. Ointment orcream formulations are also suitable for rectal or vaginal delivery,along with other standard formulations, such as suppositories. Theappropriate formulations for topical, vaginal or rectal administrationare well known to medicinal chemists.

[0058] The present invention will be of particular utility for topicalor mucosal administrations to treat a variety of bacterial infections orbacterially related undesirable conditions. Some representative examplesof these uses include treatment of (1) conjunctivitis, caused byHaemophilus sp., and corneal ulcers, caused by Pseudomonas aeruginosa;(2) otititis externa, caused by Pseudomonas aeruginosa; (3) chronicsinusitis, caused by many Gram-positive cocci and Gram-negative rods,and for general decontamination of bronchii; (4) cystic fibrosis,associated with Pseudomonas aeruginosa; (5) enteritis, caused byHelicobacter pylori (ulcers), Escherichia coli, Salmonella typhimurium,Campylobacter and Shigella sp.; (6) open wounds, both surgical andnon-surgical, as a prophylactic measure for many species; (7) burns toeliminate Pseudomonas aeruginosa or other Gram-negative pathogens; (8)acne, caused by Propionobacter acnes; (9) nose and skin infectionscaused by methicillin resistant Staphylococcus aureus (MSRA); (10) bodyodor caused mainly by Gram-positive anaerobic bacteria (i.e., use indeodorants); (11) bacterial vaginosis associated with Gardnerellavaginalis and other anaerobes; and (12) gingivitis and/or tooth decaycaused by various organisms.

[0059] In other embodiments, the antimicrobials of the present inventionfind application in the treatment of surfaces for the removal orattenuation of unwanted bacterial. For example, surfaces that may beused in invasive treatments such as surgery, catheterization and thelike may be treated to prevent infection of a subject by bacterialcontaminants on the surface. It is contemplated that the methods andcompositions of the present invention may be used to treat numeroussurfaces, objects, materials and the like (e.g., medical or first aidequipment, nursery and kitchen equipment and surfaces) to controlbacterial contamination thereon.

[0060] Pharmaceutical preparations comprising the donor bacteria areformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form, as used herein, refers to a physicallydiscrete unit of the pharmaceutical preparation appropriate for thepatient undergoing treatment. Each dosage should contain a quantity ofthe donor bacteria calculated to produce the desired antibacterialeffect in association with the selected pharmaceutical carrier.Procedures for determining the appropriate dosage unit are well known tothose skilled in the art.

[0061] Dosage units may be proportionately increased or decreased basedon the weight of the patient. Appropriate concentrations for achievingeradication of pathogenic bacteria in a target cell population or tissuemay be determined by dosage concentration curve calculations, as knownin the art.

[0062] Other uses for the donor bacteria of the invention are alsocontemplated. These include a variety agricultural, horticultural,environmental and food processing applications. For example, inagriculture and horticulture, various plant pathogenic bacteria may betargeted in order to minimize plant disease. One example of a plantpathogen suitable for targeting is Erwinia amylovora, the causal agentof fire blight. Similar strategies may be utilized to reduce or preventwilting of cut flowers.

[0063] In veterinary or animal agriculture, the killer plasmid systemsof the invention may be incorporated into animal feed (chicken, cattle)to reduce bio-burden or to eliminate certain pathogenic organisms (e.g.,Salmonella). In other embodiments, the invention may be utilized on meator other foods to eliminate unwanted or pathogenic bacteria (e.g., E.coli O157:H7 on meat, or Proteus spp., one cause of “fishy odor” onseafood).

[0064] Environmental utilities comprise, for example, engineeringBacillus thurengiensis and one of its conjugative plasmids to deliverand conditionally express insecticidal agents (e.g., for the control ofmosquitos that disseminate malaria or West Nile virus). In suchapplications, as well as in the agricultural and horticulturalapplications described above, formulation of the killer plasmids anddonor bacteria as solutions, aerosols, or gel capsules are contemplated.

[0065] In preferred embodiments of the present invention, certainfeatures are employed in the plasmids and donor cells of the inventionto minimize potential risks associated with the use of DNA orgenetically modified organisms in the environment. For instance, inenvironmentally sensitive circumstances it is preferable to utilizenon-self-transmissible plasmids. Instead, the plasmids will bemobilizable by conjugative machinery but will not be self-transmissible.As discussed hereinabove, this may be accomplished in some embodimentsby integrating into the host chromosome all tra genes whose products arenecessary for the assembly of conjugative machinery. In suchembodiments, killer plasmids are configured to possess only an origin oftransfer (oriT). This feature prevents the recipient, before or evenafter it dies, from transferring the killer plasmid further.

[0066] Another biosafety feature comprises utilizing conjugation systemswith pre-determined host-ranges. As discussed above, certain elementsare known to function only in few related bacteria (narrow-host-range)and others are known to function in many unrelated bacteria(broad-host-range or promiscuous) (del Solar et al., Mol. Microbiol.32:661-666, 1996; Zatyka and Thomas, FEMS Microbiol. Rev. 21:291-319,1998). Also, many of those conjugation systems can function in eithergram-positive or gram-negative bacteria but generally not in both (delSolar, 1996, supra; Zatyka and Thomas, 1998, supra).

[0067] Also as discussed in detail above, inadvertant proliferation ofantibiotic resistance is minimized in this invention by avoiding the useof antibiotic resistance markers. In a preferred alternative approach,the gene responsible for the synthesis of an amino acid (i.e. serine)can be mutated, generating the requirement for this amino acid in thedonor. Such mutant bacteria will prosper on media lacking serineprovided that they contain a plasmid with the ser gene whose product isneeded for growth: Thus, the invention contemplates the advantageous useof plasmids containing the ser gene or one of many other nutritionalgenetic markers. These markers will permit selection and maintenance ofthe killer plasmids in donor cells.

[0068] Another biosafety approach comprises the use ofrestriction-modification systems to modulate the host range of killerplasmids. Conjugation and plasmid establishment are expected to occurmore frequently between taxonomically related species in which plasmidcan evade restriction systems and replicate. Type II restrictionendonucleases make a double-strand break within or near a specificrecognition sequence of duplex DNA. Cognate modification enzymes canmethylate the same sequence and protect it from cleavage.Restriction-modification systems (RM) are ubiquitous in bacteria andarchaebacteria but are absent in eukaryotes. Some of RM systems areplasmid-encoded, while others are on the bacterial chromosome (Robertsand Macelis, Nucl. Acids Res. 24: 223-235, 1998). Restriction enzymescleave foreign DNA such as viral or plasmid DNA when this DNA has notbeen modified by the appropriate modification enzyme. In this way, cellsare protected from invasion of foreign DNA. Thus, by using a donorstrain producing one or more methylases, cleavage by one or morerestriction enzymes could be evaded. Site-directed mutagenesis is usedto produce plasmid DNA that is either devoid of specific restrictionsites or that comprises new sites, protecting or making plasmid DNAvulnerable, respectively against endonucleases. Broad-host rangeplasmids (eg. RP4) may evade restriction systems simply by not havingmany of the restriction cleavage sites that are typically present onnarrow-host plasmids (Willkins et al., 1996, J. Mol. Biol 258, 447-456).

[0069] Preferred embodiments of the present invention also utilizeenvironmentally safe bacteria as donors. For example, delivery of DNAvaccines by attenuated intracellular gram-positive and gram-negativebacteria has been reported (Dietrich et al., 2001 Vaccine 19, 2506-2512;Grillot-Courvalin et al., 1999 Current Opinion in Biotech 10, 477-481).In addition, the donor stain can be one of thousands of harmlessbacteria that colonize the non-sterile parts of the body (e.g., skin,gastrointestinal, urogenital, mouth, nasal passages, throat and upperairway systems). Examples of preferred donor bacterial species are setforth hereinabove.

[0070] In another strategy, non-dividing, non-growing donors areutilized instead of living cells. As discussed above, minicells andmaxicells are well studied model systems of metabolically active butnonviable bacterial cells. Minicells lack chromosomal DNA and aregenerated by special mutant cells that undergo cell division without DNAreplication. If the cell contains a multicopy plasmid, many of theminicells will contain plasmids. Minicells neither divide nor grow.However, minicells that possess conjugative plasmids are capable ofconjugal replication and transfer of plasmid DNA to living recipientcells. (Adler et al., 1970, supra; Frazer and Curtiss, 1975, supra; U.S.Pat. No. 4,968,619, supra).

[0071] Maxicells can be obtained from a strain of E. coli that carriesmutations in the key DNA repair pathways (recA, uvrA and phr). Becausemaxicells lack so many DNA repair functions, they die upon exposure tolow doses of UV. Importantly, plasmid molecules (e.g., pBR322) that donot receive an UV hit continue to replicate. Transcription andtranslation (plasmid-directed) can occur efficiently under suchconditions (Sancar et al., J. Bacteriol. 137: 692-693, 1979), and theproteins made prior to irradiation should be sufficient to sustainconjugation. This is supported by the following two observations: i)that streptomycin-killed cells remain active donors, and ii) thattransfer of conjugative plasmids can occur in the presence ofantibiotics that prevent de novo gene expression (Heineman andAnkenbauer, 1993, J. Bacteriol. 175, 583-588; Cooper and Heineman, 2000.Plasmid 43, 171-175). Accordingly, UV-treated maxicells will be able totransfer plasmid DNA to live recipients. It should also be noted thatthe conservation of recA and uvrA genes among bacteria should allowmaxicells of donor strains other than E. coli to be obtained.

[0072] Also contemplated for use in the invention are any of themodified microorganisms that cannot function because they containtemperature-sensitive mutation(s) in genes that encode for essentialcellular functions (e.g., cell wall, protein synthesis, RNA synthesis,as described, for example, in U.S. Pat. No. 4,968,619, supra). For manyapproaches, conditionally replicating killer plasmids can be used. Suchplasmids, which have been produced in accordance with the invention, canreplicate in the donor but cannot replicate in the recipient bacteriumsimply because their cognate replication initiator protein (e.g., Rep)is produced in the former cells but not the latter cells. Anothervariant plasmid contains a temperature-sensitive mutation in thementioned above rep gene, so it can replicate only at temperatures below37° C. Hence, its replication will occur in bacteria applied on skin butit will not occur if such bacteria break into the body's core.

[0073] As used herein, the term “donor cell” refers to any of theabove-listed cells, including dividing and non-dividing bacterial cells(minicells and maxicells), as well as conditionally non-functionalcells.

[0074] The following examples are set forth to describe the invention ingreater detail. They are intended to illustrate, not to limit, theinvention. Unless otherwise specified, general cloning, microbiological,biochemical and molecular biological procedures such as those set forthin Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory(1989) (“Sambrook et al.”) or Ausubel et al. (eds) Current Protocols inMolecular Biology. John Wiley & Sons (2001) (“Ausubel et al.”) are used.

EXAMPLE 1 Preparation of Runaway Replication Plasmid from Plasmid R6K

[0075] Plasmid R6K is an Escherichia coli conjugative plasmid believedto be a narrow host range. Replication of R6K derivatives containing itsoriV, called γ ori, generally requires a Rep protein, π, which isencoded by the plasmid's pir gene. The π protein is bifunctional inreplication; it acts as an activator of replication at low cellularlevels and an inhibitor of replication at elevated levels. For a reviewof R6K replication and its control by π protein, see Filutowicz &Rakowski (1998) Gene 223, 195-204.

[0076] Using site-directed mutagenesis, the inventor has obtained thefollowing three types of mutations within the pir gene:

[0077] (1) double amino acid substitution: pro106leu, phe107ser(numbering of residues according to Stalker et al. (1982) J. Mol. Biol.161:33-43).

[0078] (2) deletion of codons 106 and 107; and

[0079] (3) deletion of codons 105, 106 and 107.

[0080] The γ ori was combined with the mutated pir genes in threeconfigurations. In one configuration, the mutant gene was contained on aplasmid different from the plasmid containing the γ ori, thus providingπ protein in trans. In another configuration, the mutant gene wasintegrated into the host chromosome, thus providing π protein also intrans. In third configuration the mutant pir gene was contained on thesame plasmid with they ori, thus providing its function in cis.

EXAMPLE 2 Bacterial Cells Transformed with Plasmids Containing Mutatedpir and ori in cis are Killed

[0081]Escherichia coli cells were transformed with either (1) theplasmids containing a mutated pir gene and the γ ori in trans; or (2) aplasmid containing a mutated pir gene and the γ ori in cis.

[0082] In transformed cells containing the mutant pir and the γ ori intrans, the copy number of the γ ori plasmid was increased 20- to 25-foldin comparison to wild-type pir controls. Cells transformed with themutant pir and the γ on in cis were killed by the runaway replication ofγ ori. The occurrence of the runaway phenotype when mutant pir is in cisto the ori but not in trans is believed to be caused by the enhancedeffect of the origin activation and translation of nascent π proteinoccurring next to each other.

EXAMPLE 3 Preparation of Runaway Replication Plasmid from Plasmid RK2

[0083] Plasmid RK2 is a promiscuous plasmid that can replicate in 29(and probably many more) gram-negative species (Guiney and Lanka, 1989,p 27-54. In C. M. Thomas (ed) Promiscous plasmids in gram-negativebacteria. London, Ltd London United Kingdom.). Plasmid RK2 is a 60-kbself-transmissible plasmid with a complete nucleotide sequence known(Pansegrau et al., 1994, J. Mol. Biol. 239, 623-663). A minimal repliconderived from this large plasmid has been obtained that is devoid of allits genes except for a trfA gene, that encodes plasmid's Rep proteincalled TrfA, and an origin of vegetative replication oriV For a reviewof RK2 replication and its control by TrfA protein, see Helinski et al.,1996 (In Escherichia coli and Salmonella Cellular and Molecular Biology,Vol. 2 (ed. F. Neidhardt, et al., 2295-2324, ASM Press, WashingtonD.C.). Combinations of specific mutations in the rep gene of plasmid RK2(trfA) confer run-away replication on the minimal, self-replicatingplasmid derivatives (Haugan et al., 1995, Plasmid 33, 27-39; Toukdarianand Helinski, 1998, Gene 223, 205-211). Such plasmids elicit a killingeffect when introduced into wild type E. coli strains by transformationor electroporation. The inventors laboratory also constructed a plasmidwhich can inflict killing on bacterial host conditionally. This wasachieved by using an inducible promoter which governs expression of ahyperactive version of trfA (trfA264 267); in the absence of an inducer,plasmid copy number is low (harmless) but in the presence of the inducerrun-away replication occures, killing the host. The run-away plasmids,both constitutive and conditional, can be maintained in speciallyconstructed strains in which a wild-type allele of the trfA gene(providing replication repressor) is also present, thereby suppressingover-replication (killing) by complementation. This and the previousexamples illustrate not only the use of R6K derivatives to kill unwantedbacteria (Example 2 above) but also specifically constructed derivativesof other plasmids such as RK2.

[0084] The present invention is not limited to the embodiments describedand exemplified above, but is capable of variation and modificationwithout departure from the scope of the appended claims.

I claim:
 1. An antimicrobial agent, which comprises a donor cellharboring at least one transmissible plasmid comprising: a) an origin ofreplication for synthesizing the plasmid in the donor cell, whereininitiation of replication at the origin is negatively controlled by aplasmid replication repressor; b) an origin of transfer from whichconjugative transfer of the transmissible plasmid initiates from thedonor cell to at least one recipient cell; and, optionally, c) at leastone selectable marker gene; wherein the donor cell further comprises oneor more transfer genes conferring upon the donor cell the ability toconjugatively transfer the transmissible plasmid to the recipient cell,and wherein the donor cell produces the plasmid replication repressor,and further wherein the at least one recipient cell is a pathogenicbacterium that does not produce the plasmid replication repressor. 2.The antimicrobial agent of claim 1, wherein the transfer genes arecontained on a helper plasmid within the donor cell, such that thetransmissible plasmid is transmissible from the donor cell to arecipient cell, but is not further self-transmissible from the recipientcell to another recipient cell.
 3. The antimicrobial agent of claim 1,wherein the transfer genes are contained on the transmissible plasmid,such that the transmissible plasmid is self-transmissible from the donorcell to a recipient cell, and further from the recipient cell to anotherrecipient cell.
 4. The antimicrobial agent of claim 1, win thetransmissible plasmid comprises a derivative of a naturally-occurringtransmissible plasmid containing a gene encoding the replicationrepressor that has been mutated to produce a non-functional replicationrepressor.
 5. The antimicrobial agent of claim 4, wherein thenaturally-occurring transmissible plasmid is selected from the groupconsisting of RK2, R6K, pCU1, p15A, pIP501, pAM 1 and pCRG1600.
 6. Theantimicrobial agent of claim 5, wherein the naturally-occurring plasmidis R6K and the mutation comprises a mutation in the R6K pir gene suchthat its encoded protein comprises an at least one amino acid deletionor substitution at amino acids 105, 106 or
 107. 7. The antimicrobialagent of claim 1, wherein the donor cell is selected from the groupconsisting of dividing bacteria, maxicells, minicells and conditionallynon-functional bacteria.
 8. The antimicrobial agent of claim 7, whereinthe donor cell is a non-pathogenic strain of bacteria selected from thegroup consisting of Escherichia coli, Lactobacillus spp., Lactococcus,Bifidobacteria, Eubacteria, and bacterial minicells.
 9. Theantimicrobial agent of claim 1, wherein the recipient cell is apathogenic strain of bacterium selected from the group consisting ofCampylobacter spp., Enterobacter. spp., Enterococcus spp., Escherichiacoli, Gardnerella vaginalis, Haemophilus spp., Helicobacter pylorii,Mycobacterium tuberculosis, Propionobacter acnes, Pseudomonas aeruginosaand other Pseudomonas spp., Salmonella typhimurium, Shigella spp. andStaphylococcus spp.
 10. The antimicrobial agent of claim 1, wherein theorigin of replication is derived from a plasmid selected from the groupconsisting of R6K, RK2, rts1, p15A and RSF1010.
 11. The antimicrobialagent of claim 1, wherein the origin of replication is selected from thegroup consisting of F and P1.
 12. The antimicrobial agent of claim 1,wherein the selectable marker gene confers a nutritional selectionadvantage on cells containing the transmissible plasmid.
 13. Theantimicrobial agent of claim 1, wherein the transfer genes are derivedfrom a plasmid selected from the group consisting of F, R6K and Ti. 14.A pharmaceutical preparation for reducing or eliminating a targetpopulation of bacteria in a subject, comprising the antimicrobial agentof claim 1 formulated for a pre-determined route of administration tothe subject.
 15. The pharmaceutical preparation of claim 14, wherein thepre-determined route of administration is selected from the groupconsisting of: topical, oral, nasal, pulmonary, ophthalmic, aural,rectal, urogenital, subcutaneous, intraperitoneal and intravenous.
 16. Amethod of reducing or eliminating a target population of bacteria in asubject, the method comprising administering to the subject theantimicrobial agent of claim 1 in a manner such that the donor cells ofthe antimicrobial agent come into conjugative proximity to the targetbacterial cells, such that the transmissible plasmids of the donor cellsare transferred from the donor cells to the target bacterial cells,whereupon the transmissible plasmids commence unchecked replication,thereby killing the target bacterial cells.
 17. The method of claim 16,wherein the subject is a human or an animal.
 18. The method of claim 16,wherein the subject is a plant.
 19. The method of claim 16, wherein thesubject is food or feed.
 20. An antimicrobial agent, which comprises adonor cell harboring at least one transmissible plasmid comprising: a)an origin of replication for synthesizing the plasmid in the donor cell;b) an origin of transfer from which conjugative transfer of thetransmissible plasmid initiates from the donor cell to at least onerecipient cell; c) at least one killer gene that, upon expression in abacterial cell, produces a product that kills the cell; and, optionally,d) at least one selectable marker gene; wherein the donor cell furthercomprises one or more transfer genes conferring upon the donor cell theability to conjugatively transfer the transmissible plasmid to therecipient cell, and wherein the donor cell is modified so as to beunaffected by the product of the killer gene, and further wherein the atleast one recipient cell has not been modified so as to be unaffected bythe product of the killer gene.
 21. The antimicrobial agent of claim 20,wherein the transfer genes are contained on a helper plasmid within thedonor cell, such that the transmissible plasmid is transmissible fromthe donor cell to a recipient cell, but is not furtherself-transmissible from the recipient cell to another recipient cell.22. The antimicrobial agent of claim 20, wherein the transfer genes arecontained on the transmissible plasmid, such that the transmissibleplasmid is self-transmissible from the donor cell to a recipient cell,and further from the recipient cell to another recipient cell.
 23. Theantimicrobial agent of claim 20, wherein the killer gene kills the cellsby being expressed and thereby producing a gene product that isdetrimental or lethal to bacterial cells, and the donor cells have beenmodified so as to repress the expression of the killer gene, therebyavoiding production of the detrimental or lethal gene product.
 24. Theantimicrobial agent of claim 20, wherein the killer gene is obtainedfrom a bacteriophage.
 25. The antimicrobial agent of claim 24, whereinthe bacteriophage is selected from the group consisting of T-seriesphages, P1, p22 and λ.
 26. The antimicrobial agent of claim 20, whereinthe donor cell is selected from the group consisting of dividingbacteria, maxicells, minicells and conditionally non-functionalbacteria.
 27. The antimicrobial agent of claim 26, wherein the donorcell is a non-pathogenic strain of bacteria selected from the groupconsisting of Escherichia coli, Lactobacillus spp., Lactococcus,Bifidobacteria, Eubacteria, and bacterial minicells.
 28. Theantimicrobial agent of claim 20, wherein the recipient cell is apathogenic strain of bacterium selected from the group Campylobacterspp., Enterobacter spp., Enterococcus spp., Escherichia coli,Gardnerella vaginalis, Haemophilus spp., Helicobacter pylorii,Mycobacterium tuberculosis, Propionobacter acnes, Pseudomonas aeruginosaand other Pseudomonas spp., Salmonella typhimurium, Shigella spp. andStaphylococcus spp.
 29. The antimicrobial agent of claim 20, wherein theorigin of replication is derived from a plasmid selected from a thegroup consisting of R6K, RK2, rts1, p15A and RSF1010.
 30. Theantimicrobial agent of claim 20, wherein the origin of replication isselected from the group consisting of F and P1.
 31. The antimicrobialagent of claim 20, wherein the selectable marker gene confers anutritional selection advantage on cells containing the transmissibleplasmid.
 32. The antimicrobial agent of claim 20, wherein the transfergenes are derived from a plasmid selected from the group consisting ofF, R6K and Ti.
 33. A pharmaceutical preparation for reducing oreliminating a target population of bacteria in a subject, comprising theantimicrobial agent of claim 20 formulated for a predetermined route ofadministration to the subject.
 34. The pharmaceutical preparation ofclaim 33, wherein the predetermined route of administration is selectedfrom the group consisting of: topical, oral, nasal, pulmonary,ophthalmic, aural, rectal, urogenital subcutaneous, intraperitoneal andintravenous.
 35. A method of reducing or eliminating a target populationof bacteria in a subject, the method comprising administering to thesubject the antimicrobial agent of claim 20 in a manner such that thedonor cells of the antimicrobial agent come into conjugative proximityto the target bacterial cells, such that the transmissible plasmids ofthe donor cells are transferred from the donor cells to the targetbacterial cells, whereupon the at least one killer gene is expressed,thereby producing the product that kills the target bacterial cells. 36.The method of claim 35, wherein the subject is a human or an animal. 37.The method of claim 35, wherein the subject is a plant.
 38. The methodof claim 35, wherein the subject is food or feed.